Patch Clamp Techniques 1&2 Flashcards
What is resistance
a measure of the opposition to current flow in an electrical circuit.
What is current
Rate of flow of charged particles aka the charge transfered over time
What is voltage
The electrical potential difference - no current if voltage the same at both ends to need a difference between the ends.
E.g. 10V at one end 0V at the other, will flow towards 0V
What is ground
A point of reference - no change in voltage but different starting point e.g. 20mV instead of 0 so inside the cell was 60mV but with ground included its 40mV
What is the relationship between current resistance and flow
All rely on each other so -
V=IR
What is capacitance
The change in potential (charge)
Units - farads
What is the role of a capacitors
A capacitor stores and releases current in a circuit - can release current when no voltage if the energy is stored
What has capacitance in cells
Cell membrane
What has current in cells
Ions flowing through cell membrane
What has voltage in cells
The potential difference across the membrane
What has resistance in cells
Ion channels
Does the number of channels (resistors) in parallel effect the current
No - current spread over 1 or 20 resistors, it doesnt matter, all add up to total current
It = I1 + I2 + I3 etc
What is the relationship with conductance and resistance
Conductance is the inverse of resistance
What happens at the ion equilibrium potential
There is no net flow of ions across the membrane - it is different for different ions
What two factors allows the cell to have a membrane potential
Concentration gradient is due to ion pumps (Na/K ATPase)
Electrical potential is due to selective membrane permeability
What is the resting potential of neuron cells
- 60mV
What is the resting potential of muscle cells
- 90mV
Where is there more potassium
Inside the cell
Where is there more sodium
Outside the cell
Where is there more chloride
Outside the cell
Where is there more calcium
Outside the cell
What is the purpose of the voltage clamp
Allows the voltage across the membrane to be fixed and this allows the current flowing across that membrane to be measured.
This allowed analysis of single ion channels as allowed control of the external and internal cell environments and recorded low noise (single-channel currents)
How does the patch clamp technique work
Isolates a patch of membrane (pinches) from the external solution with a glass pipette and record current flowing through the patch (a single channels current)
What is important about the patch seal resistance
Needs to be tight. Therefore the seal resistance must be greater than the pipette to stop current flowing outwith patch as the true isolated current will not be obtained if allowed to escape.
What does a loose seal give
Escaped current
AND
The background noise
What is a gigaseal
Mechanically stable high resistance seal which ensures as much current as possible enters the glass pipette
How are gigaseals made
Suction to the membrane of the cell increases the seal - gives gigaseal
Allows even the smallest of currents to be obtained
Give the 4 types of patch-clamp gigaseal
Cell attached - suction of patch but patch still attached to cell
Inside out patch - suction of patch so much that it tears away from cell that the inside of the cell is present in the glass pipette
Whole cell recording - suction of patch is removed and make recordings for the whole of the inside of a cell
Outside-out patch - suction of patch so much that it tears away from cell that the outside is present in the glass pipette
What was found with ion channels using patch clamp techniques
Clamps are always opening and closing
Its instantaneous
Modulators
What are the advantages of using cells for patch clamps
Easier access
Can be used to express specific proteins and their effects
Better space clamp - amplifier can apply the voltage quite well across the membrane unlike the complexity of tissue and multiple cells which is harder
What are the advantages of using tissue sections for patch clamps
Connectivity is preserved
No culture media - so no added solutions which may not be physiological
What is the main purpose of the solution the cells are in
Mimics the composition of the fluid in vivo (extracellular and pipette solution - intracellular)
What is the purpose of a vibratome
Has a vibrating blade and tissue holder to cut the tissue sections
What is the purpose of a pipette puller
Has a heating element and glass capillary holder.
This heats the glass and produces a narrow tip which is the pipette.
What is the purpose of the amplifier
Maintains a constant membrane potential and is attached to the pipette
How do you know that you are clamping the right area
Has pipette holder with a micromanipulator and microscope to specifically target the correct channel
How does patching occur
Clear flow of liquid outside of the pipette with air to allow clean contact with the cell membrane.
Can measure current and knows the voltage we are applying using amplifier so we can tell with V=IR the resistance of the pipette to see if the pipette is sufficient (Gigapipette)
How do we know if the micropipette is touching the cell
Using V=IR –>
Know constant voltage by amplifier
Decrease in current and increase in resistance shows that it is attached.
What happens to current when patch suction occurs
Current decreases from very high to tiny as removal of background noise and left with single channel current. Also shows that voltage is very high (desired as gigapipette)
How does whole cell recording work
More section occurs until the cell membrane breaks, this causes a drop in current which is then compensated which shows the whole cell current
What is the difference between voltage clamps and current clamps
Voltage clamp - set voltage, measures current (e.g. post-synaptic currents)
Current clamp - set current, measures voltage
(e.g. action potentials)
Why are cell-attached recordings important
Can look at changes across the membrane without disturbing the whole cell
Can whole cell patch clamp be done in vivo
Yes and vitro
How can patch clamp help in pharmacology
Tool to investigate cell mechanism using well studied drug
Tool to investigate drug mechanism using well studied cell
How can whole cell recordings be used in post-synaptic currents
Patch on input cell for whole cell recording and the currents can be recorded.
Stimulus - specific stimulus to pre-synaptic neuron and see its effects in post-synaptic cell
Known inhibitor of pre-synaptic neuron and see its effects on post-synaptic cell (see spontaneous actions of post-synaptic neuron independent of pre-synaptic)
Use drugs to block channels and assess its effects
How does patch clamp techniques and optogentics work together
Channelrhodopsin (light sensitive cation channel) opens to blue light.
Current can be recorded via optogenetics in the channelrhodopsin neuron or its post-synaptic neuron (channelrhodopsin-assisted circuit mapping - CRACM)
How do automated patch clamp systems work
WIDER READING - Vasilyev et al 2005
Collects large amounts of data quickly over a shorter time than traditional patch clamp techniques.
This technique is when cells are placed on an injection molded cast chip which cells attach to. This substitutes the micropipette and has an integraded electrode for controlling voltage or current.
This allows cells to be inside out or whole cell patch clamp recordings.
This allows different solution wells to be applied to the cells to look at their reactions.
What is electrode fabrication and why is it important
WIDER READING - (Kornreich, 2007)
The formation of the micropipette by melting it with an electrode.
To ensure a tight seal with no impurities - all solutions are flitered via a micronfilter before experiments and electrode fabrication of micropipettes are done as close as possible to the time they are being used and then kept in sealed chamber to prevent airborne particles from contaminating the tips.
What kind of glass is used for the micropipette and why
WIDER READING - (Kornreich, 2007)
Borosilicate glass - low softening temp to make the pipette, low electrical noise, forms high resistance seals easily.
What is two-photon targeted patching (TPTP) and what did it discover in parvalbumin-EGFP positive interneurons in the somatosensory cortex.
WIDER READING - Margrie et al 2003
Uses two-photon imaging (imaging used for dense tissue with penetration of 1mm) to guide in vivo whole-cell recordings of labelled cortical parvalbumin-EGFP positive interneurons in the somatosensory cortex.
Showed that spontaneous and evoked spikelets (spike depolarisations) are needed to be synchronized to terminate episodes of strong neuronal network excitation.
What is an advantage of the cell-attached current clamp over whole-cell recording
WIDE READING - Perkins 2006
An advantage of cell-attached current-clamp over whole-cell recording is that it accurately depicts whether a synaptic potential is hyperpolarizing or depolarizing without the risk of changing its polarity.
What was obtained regarding cardiomyocytes and patch-clamp techniques?
WIDER READING - Verkerk et al 2021
Hard to test drugs on cardiomyocytes as frequently depolarized state when isolated.
Did a dynamic clamp of providing an inward rectifier K current to form a non-depolarized resting membrane potential.
This allowed the testing of multiple drugs - lidoaine, nifedipine etc) which evoked APs so showed it worked.
Therefore, dynamic clamp represents a promising tool for testing novel antiarrhythmic drugs.
Why are sperm difficult to test patch-clamp techniques on
WIDER READING - Lishkko et al 2014
Tight, small cell membrane with lack of cytoplasm so hard to get gigaseal with micropipette.
However, found a region on the plasma membrane called the cytoplasmic droplet that is loosely attached to the rigid intracellular structures and with which a tight gigaohm seal can be formed with the patch pipette
Using patch-clamp testing, what was discovered about sperm motility
WIDER READING - Kirichok et al 2006
Calcium and alkali dependent - Sperm flagella become hyperactivated due to an influx of calcium via CatSper1 channel because of intracellular alkalization (discovered via patch clamp) which causes the hypermobility.
Using a whole-cell patch-clamp, what was found regarding malaria and its effects on RBCs
WIDER READING - Bouyer et al 2007
Did whole-cell patch clamp testing on RBCs infected with malaria parasite plasmodium falciparum in physiological ion concentrations.
Shown that the membrane current is 100-150 fold higher than in uninfected RBCs compared to infected cells.
This was because - the inwardly rectifying anion channel and Ps small conductance anion channel have enhanced activity in these infected cells. Shown that erythrocyte permeability is increased by the parasite. Proven with whole cell patch clamp.
What did patch clamp techniques show regarding diclofenac in mice airways
WIDER READING - Chen et al 2019
Diclofenac relaxes the smooth muscle in the tracheal by relaxing high potassium and Ach contraction of the tracheal rings via decreasing ctosolic calcium in the airway smooth muscle cells through inhibiting voltage gated calcium channels preventing calcium influx which prevents contraciton.
Suggests - diclofenac may be good as a antibronchospasmic drug used in treating respiratory diseases such as asthma and chronic obstructive pulmonary disease.
